Optimizing Primer Design in Restriction and Ligation-Independent Polymerase Chain Reaction (pcr) Cloning

This application claims priority to and the benefit of U.S. Provisional Application No. 63/569,930, filed Mar. 26, 2024, which is hereby incorporated herein by reference in its entirety.

This application contains a sequence listing filed in ST.26 format entitled “322203-1140 Sequences” created on Jun. 4, 2025 and having 41,763 bytes. The content of the sequence listing is incorporated herein in its entirety.

Molecular cloning, a fundamental pillar of diverse biological research, often involves laborious multi-step procedures demanding significant time and resources. In a standard molecular cloning process, deoxyribonucleic acid (DNA) from an organism of interest is obtained and cleaved at specific site through the use of various enzymes. Once fragmented, the DNA is recombined with a cloning vector to produce recombinant DNA. The recombinant DNA can be introduced into a host organism to be reproduced or cloned.

Disclosed are various approaches for optimizing primer design in restriction and ligation-independent polymerase chain reaction (PCR) cloning. FastCloning is a widely-used cloning technique for plasmid reconstruction, allowing for a greatly simplified method for inserting any DNA fragment into a plasmid vector or into a gene. This paradigm shift in PCR cloning, has streamlined the process by eliminating laborious, multi-step traditional methods. FastCloning utilizes overlapping PCR primers and DpnI digestion for seamless integration of insert DNA into any desired vector position, regardless of restriction sites. This versatility makes FastCloning ideal for constructing fusion proteins, chimeric cDNAs, and manipulating genes with unparalleled ease.

However, efficient primer design can be a significant hurdle, particularly for newcomers, as errors can lead to failed cloning attempts. To address this bottleneck, this disclosure presents FastCloneAssist, a user-friendly Python™ program that can automate FastCloning primer design with minimal user input. Users simply provide a vector and insert sequences, along with the desired melting temperature (Tm), and FastCloneAssist can calculate the optimal primer parameters for efficient amplification and seamless integration using established bioinformatics libraries. This tool can simplify and accelerate FastCloning, making this a powerful technique accessible to researchers of all levels and expediting scientific discovery.

Molecular cloning can often involve laborious multi-step procedures demanding significant time and resources. Fortunately, FastCloning emerged as a transformative PCR-based approach, eliminating the need for restriction enzymes and ligation while fostering rapid fragment integration. This method relies on specifically designed primers for amplifying both vector and insert DNA, followed by DpnI treatment to selectively digest parental templates, and facilitate in vivo ligation (mechanism remains under investigation).

While FastCloning offers undeniable advantages, meticulous primer design remains crucial for successful amplification and seamless integration of the target fragment. Traditional strategies can often involve laborious manual calculations and sequence adjustments to attain optimal primer Tm, length, and overlap sequences, with a single base error potentially jeopardizing the entire cloning project. Moreover, existing online tools often lack customization options and can require coding expertise, limiting their accessibility to novice users.

To address these challenges, this disclosure introduces FastCloneAssist, a user-friendly Python™ script designed to streamline FastCloning primer design. Requiring minimal user input (vector and insert sequences), FastCloneAssist can automate the selection of optimal primer lengths, melting temperatures, and overlap sequences, ensuring efficient amplification and in vivo ligation.

Among other features and benefits of the present disclosure, FastCloneAssist can provide automated optimization for optimizing primer lengths, melting temperatures, and overlap sequences based at least in part on user-provided sequences. This can eliminate tedious manual calculations. Additionally, FastCloneAssist offers both default and user-defined Tm options, allowing for flexibility in primer design. Another benefit of the system is that it can require no coding knowledge, making it accessible to users of all skill levels. The platform independence of this invention also means that it can run easily on local computers in the Python™ environment and in the cloud. By streamlining primer design and overcoming technical barriers, FastCloneAssist can empower researchers to leverage the full potential of FastCloning, potentially accelerating diverse molecular biology applications.

In the following discussion, a general description of the system and its components is provided, followed by a discussion of the operation of the same. Although the following discussion provides illustrative examples of the operation of various components of the present disclosure, the use of the following illustrative examples does not exclude other implementations that are consistent with the principles disclosed by the following illustrative examples.

The scheme of primer designing is shown in. The FastCloneAssist basic method can be divided into two classes. The first method is Class 1, which generates primer pairs for a chimera creation where an insert DNA (>100 bp) will be PCR amplified and inserted into a PCR linearized vector DNA. Class 1 method generates a total of two pairs of primers, one pair for vector and one for insert DNA. The second method is Class 2, which generates a primer pair which can be used to delete a region or insert or delete and insert. According to some examples, the insert should be less than 120 bp. Both Class 1 and Class 2 will be described in further detail below.

In some examples, the tool described herein uses the following libraries for the listed functions:

To begin, a user can provide one or more inputs to the system. A first input can be a sequence. In some embodiments, for class 1, a user can provide a sequence of their desired chimera arrangement. For example, the user can provide the sequence in the format: vector_part-1 (5′ side to the insert's vector sequence [>40 bp])+insert sequence+vector_part-2 (3′ side to the insert's vector sequence [>40 bp])). According to various examples, these three pieces of the sequence must be separated or marked by “+” symbol (e.g., vector part 1+insert+vector part 2). According to various examples, the “vector part 1” and “vector part 2” should not be less that 40 bp. In some embodiments, the “vector part 1” and “vector part 2” should not be less than 50 bp, 70 bp, 100 bp, or 150 bp. Below is an example input sequence format for Class 1 (SEQ ID NO: 35):

The insert sequence is in lower case and joined by “+” symbols into the vector part 1 and vector part 2 at the 5′ end and 3′ end of the insert sequence respectively.

In some embodiments, the user may also be asked to input a choice to customize the desired Tm range for primer design. In some embodiments, the system has a default range of, for example, approximately 55-65° C.

After receiving user inputs, the system can begin sequence processing. In some embodiments, the script splits the input sequence into specific parts as marked by the symbols (e.g., “+”). The system can identify and remove any gaps or line breaks from the sequences. Then, in some embodiments, the system can generate protein translation into one or more frames (e.g., −3, −2, −1, +1, +2, +3, etc.) for the final construct sequence.

Next, the system conducts a primer design step. In this step, the system can calculate the initial Tm values for potential primer sequences based at least in part on their nucleotide content. Then, the system can adjust primer lengths iteratively to achieve Tm values within the desired range (e.g., either the default or user-specified range). The system can determine the appropriate locations for primers based at least in part on the boundaries of the vector parts and insert.

In some embodiments, the system can design primers in Class 1 or in Class 2. To design primers for the vector amplification in Class 1, “vector_part1” and “vector_part2” sequences can be used. The “vector_part1” region specific primer can be designed by selecting nucleotides from 3′ ends toward 5′ of the sequence and the primer length adjusted to meet the desired Tm using SeqUtils modules from Biopython. The reverse complement of this sequence can be used as the reverse primer for vector. Forward primer for the vector can come from the “vector_part2” nucleotides which get selected from 5′ region of the sequence to meet the Tm and length of primer. Primers for the insert sequence can come from the 5′ and 3′ terminals of the insert sequence and get a 16 bp complementary overhang from respective vector regions. The forward primer of the insert (FPI) get overhang from vector_part1 whereas reverse primer of the insert (RPI) get overhang from the vector_part2, see the. The length of FPI and RPI (without their overhangs) also get adjusted to meet the desired Tm values.

For Class 2 primer design, the system can design primers for deletion, insertion and/or deletion & insertion. In some embodiments, a user is asked to provide DNA sequences in a format where the deletion region (start & end) will be marked by “*” symbols and the insert sequence will be provided at the end of sequence marked by another “*” symbol. One example, demonstrating the format of the input, is provided below (SEQ ID NO: 36):

The deletion region is shown underlined and marked by “*” at the start and finish. Likewise, the insert sequence, in small case, is demarked by another “*” symbol.

In some embodiments, a user may wish to only make an insertion, without a deletion. In such examples, the user is asked to provide an input sequence by marking the insertion point by two stars “**” or another symbol, and to provide the insert sequence as above. One example, demonstrating the format of the input sequence, is shown below (SEQ ID NO: 37):

For deletion-only primer designing, the input sequence format can be as follows: the deletion can be marked using a star (*) or other symbol, as described in the deletion insertion example. Further, the format can include placement of a third star at the end of sequence. One example, demonstrating the format of the deletion-only input sequence, is shown below (SEQ ID NO: 38):

In some embodiments, the system can produce one or more outputs. The system can generate and display a FASTA format of the final construct sequence. In some embodiments, the system generates and displays translations in all three frames (for example, when the optional translation feature is used). In addition, according to various examples, the system can output designed primer sequences, along with their Tm values and lengths. In some examples, the system can output a report saved as .txt file or other file format.

The FastCloneAssist tool can be run in a python environment installed locally. In some embodiments, a user can utilize the FastCloneAssist script by first downloading it from a GitHub repository and executing it using Python. According to various examples, the code is structured into three parts. A user can begin by running the first part of the code to install the required libraries (e.g., Biopython & primer3). In some embodiments, it is important to note that the first part only needs to be executed once for a system, as it installs the necessary libraries. Once this step is completed, the user can proceed to run the second part. The second part of the code can import all required modules from the installed libraries. Note the tkinter, sys, and os modules can be imported from the default Python installation.

Running the third part of the code (the primer design part), a user can be prompted to choose either Class 1 or Class 2 style of primer designing (see above). Following a selection, the next required input will be the sequence. Ensure the sequence is pre-prepared with symbols as described above for the specific class and need. The next prompts can ask the user if the user wants to choose any specific Tm or use the default range (55-65° C.)? The user can enter “no”, but if the user input is “yes,” the code will prompt the user to enter the Tm range, such as “60-64”. After this input, the script can generate a report as a text file. In some embodiments, the user will receive a popup asking the user to save the file.

In some embodiments, the FastCloneAssist tool can be run using google Colab. In such embodiments, no Python installation is required on the computing device. To use the tool in Google Colab, a user can first create a Colab account using a Google account. The user can download the Colab version of the script from a GitHub link and save it in your Google Drive. In Colab, the user can import the script from their Google Drive and run it. The user can right click on file “FastCloneAssist_Colab” file in their google drive and choose “run with Google Colaboratory.” Note, in Colab, a user may need to run the first part of the code each time they start a new session, as Google Cloud may not store the installed library once the session ends. The primer can be saved as a text file in the download folder.

While the above describes two nonlimiting examples of a user journey in using the FastCloneAssist tool, there are many other user experiences that could occur for the user to use FastCloneAssist. The tool FastCloneAssist streamlines and expedites Fast Cloning, enhancing accessibility for researchers of all levels. This advancement holds the promise of further democratizing molecular biology and accelerating the pace of scientific discovery.

With reference to, shown is a network environmentaccording to various embodiments. The network environmentcan include a computing environmentand a client device, which can be in data communication with each other via a network

The networkcan include wide area networks (WANs), local area networks (LANs), personal area networks (PANs), or a combination thereof. These networks can include wired or wireless components or a combination thereof. Wired networks can include Ethernet networks, cable networks, fiber optic networks, and telephone networks such as dial-up, digital subscriber line (DSL), and integrated services digital network (ISDN) networks. Wireless networks can include cellular networks, satellite networks, Institute of Electrical and Electronic Engineers (IEEE) 802.11 wireless networks (i.e., WI-FI®), BLUETOOTH® networks, microwave transmission networks, as well as other networks relying on radio broadcasts. The networkcan also include a combination of two or more networksExamples of networkscan include the Internet, intranets, extranets, virtual private networks (VPNs), and similar networks.

The computing environmentcan include one or more computing devices that include a processor, a memory, and/or a network interface. For example, the computing devices can be configured to perform computations on behalf of other computing devices or applications. As another example, such computing devices can host and/or provide content to other computing devices in response to requests for content.

Moreover, the computing environmentcan employ a plurality of computing devices that can be arranged in one or more server banks or computer banks or other arrangements. Such computing devices can be located in a single installation or can be distributed among many different geographical locations. For example, the computing environmentcan include a plurality of computing devices that together can include a hosted computing resource, a grid computing resource or any other distributed computing arrangement. In some cases, the computing environmentcan correspond to an elastic computing resource where the allotted capacity of processing, network, storage, or other computing-related resources can vary over time.

Various applications or other functionality can be executed in the computing environment. The components executed on the computing environmentinclude the FastCloneAssist application, and other applications, services, processes, systems, engines, or functionality not discussed in detail herein.

The FastCloneAssist applicationcan be executed to receive one or more inputs from a client device. The FastCloneAssist applicationcan be executed to receive a selection of a class, designate a format for a sequence input, receive a sequence input and process the sequence input. The FastCloneAssist applicationcan determine a plurality of constraints, such as a melting temperature, a primer length, and/or an overlap sequence. Next, the FastCloneAssist applicationcan design a primer based at least in part on the plurality of constraints.

Also, various data is stored in a data storethat is accessible to the computing environment. The data storecan be representative of a plurality of data storeswhich can include relational databases or non-relational databases such as object-oriented databases, hierarchical databases, hash tables or similar key-value data stores, as well as other data storage applications or data structures. Moreover, combinations of these databases, data storage applications, and/or data structures may be used together to provide a single, logical, data store. The data stored in the data storeis associated with the operation of the various applications or functional entities described below. This data can include sequence libraries, and potentially other data.

The sequence librariescan represent any grouping or catalog of DNA fragments which have adapters attached. Examples of sequence librariesinclude Biopython, which can provide tools for sequence manipulation and translation. In addition, sequence libraries may include the Biopython SeqUtils.MeltingTemp tool, which has specific capabilities for Tm calculations. Another example is Primer3, which can be used for primer design and Tm calculations; Bio.SeqUtils.MeltingTemp, which has specific capabilities for Tm calculations; Tkinter, which can be used to create the optional GUI interface; and many other sequence libraries, databases, or primer design tools.

The client deviceis representative of a plurality of client devices that can be coupled to the networkThe client devicecan include a processor-based system such as a computer system. Such a computer system can be embodied in the form of a personal computer (e.g., a desktop computer, a laptop computer, or similar device), a mobile computing device (e.g., personal digital assistants, cellular telephones, smartphones, web pads, tablet computer systems, music players, portable game consoles, electronic book readers, and similar devices), media playback devices (e.g., media streaming devices, BluRay® players, digital video disc (DVD) players, set-top boxes, and similar devices), a videogame console, or other devices with like capability. The client devicecan include one or more displays, such as liquid crystal displays (LCDs), gas plasma-based flat panel displays, organic light emitting diode (OLED) displays, electrophoretic ink (“E-ink”) displays, projectors, or other types of display devices. In some instances, the displaycan be a component of the client deviceor can be connected to the client devicethrough a wired or wireless connection.

The client devicecan be configured to execute various applications such as a client applicationor other applications. The client applicationcan be executed in a client deviceto access network content served up by the computing environmentor other servers, thereby rendering a user interfaceon the display. To this end, the client applicationcan include a browser, a dedicated application, or other executable, and the user interfacecan include a network page, an application screen, or other user mechanism for obtaining user input. The client devicecan be configured to execute applications beyond the client applicationsuch as email applications, social networking applications, word processors, spreadsheets, or other applications.

Moving to, shown is a flowchart that provides one example of the operation of a portion of the FastCloneAssist application. The flowchart ofprovides merely an example of the many different types of functional arrangements that can be employed to implement the operation of the depicted portion of the FastCloneAssist application. As an alternative, the flowchart ofcan be viewed as depicting an example of elements of a method implemented within the network environment.

Beginning with block, the FastCloneAssist applicationcan install one or more sequence libraries. In some embodiments, the FastCloneAssist applicationcan find and download the sequence librariesfrom a computer, server, device, or other system connected over the network. In some embodiments, the FastCloneAssist applicationcan find and download the sequence librariesfrom the internet. The FastCloneAssist applicationcan save the sequence librariesin a data storein the computing environment, or in another location.

At block, the FastCloneAssist applicationcan receive a class selection. The selection of a class can be a “1” input or a “2” input or another class selection input. In some embodiments, the FastCloneAssist applicationreceives a class selection from a client devicevia the client application. The selection of a class can come from a user interaction with the user interfaceof a client deviceand be transmitted to the FastCloneAssist applicationby the client application. In some embodiments, the FastCloneAssist applicationreceives a selection of a class from another source in the network environment.

Moving to block, the FastCloneAssist applicationcan receive a sequence input. The sequence input can be formatted according to the class selection received at block. In some embodiments, the FastCloneAssist applicationcan cause a prompt to appear on a user interfaceof a client devicein response to receiving a class selection at block. The prompt can request a sequence input in a format corresponding to the class selection received at block. The user can provide a sequence input in the corresponding format, which can be transmitted from the client deviceto the FastCloneAssist applicationby the client application. In some embodiments, the FastCloneAssist applicationreceives the sequence input from another source in the network environment.

Next, at block, the FastCloneAssist applicationcan process the sequence input received at block. In some embodiments, the FastCloneAssist applicationinterprets the sequence input according to the format corresponding to the class selection received at block. The FastCloneAssist applicationcan split the sequence input into various different parts using the designated format. Additionally, in some embodiments, the FastCloneAssist applicationcan remove spaces, gaps, line breaks, or other miscellaneous formatting errors from the sequences. According to various examples, the FastCloneAssist applicationcan generate protein translation from the sequence input into one or more frames (e.g., +1, +2, +3, etc.) for the final construct sequence.

At block, the FastCloneAssist applicationcan begin designing a primer by selecting a melting temperature (Tm). In some embodiments, the FastCloneAssist applicationcan calculate an initial melting temperature for the potential primer sequences based at least in part on the nucleotide content of the input sequence received at block. In some embodiments, the FastCloneAssist applicationselects the melting temperature based at least in part on the sequence input received at blockand based at least in part on the sequence librariesinstalled at block. In some embodiments, the FastCloneAssist applicationselects a melting temperature by generating a prompt to be presented to a user, the prompt requesting a melting temperature input. In some embodiments, the FastCloneAssist applicationselects a melting temperature based at least in part on a response to such a prompt. For example, the FastCloneAssist applicationcan select a melting temperature based at least in part on a melting temperature input. In some embodiments, the FastCloneAssist applicationcan select a melting temperature from a default range (e.g., 55-65° C.).

Moving to block, the FastCloneAssist applicationcan select a primer length. In some embodiments, the FastCloneAssist applicationcan select a primer length iteratively with selecting a melting temperature at block. For example, the FastCloneAssist applicationcan adjust the selected primer length based at least in part on whether the melting temperature corresponding to the primer length is within the desired melting temperature range. In some embodiments, the FastCloneAssist applicationcan select a primer length based at least in part on the sequence librariesinstalled at block, the class selection received at block, and/or the sequence input received at block.

Moving to block, the FastCloneAssist applicationcan select overlap sequences. The overlap sequences can be selected based at least in part on the primer sequences. As shown in& B for Class 1, the overlap sequences f1c and r1c are reverse complements of the primer sequences f1 and r1. In some embodiments, the optimized overlap sequence length is in the range of 15 to 17 base pairs (bp). In some embodiments, the optimized overlap sequence length is in the range of 14 to 18 bp. In some embodiments, the optimized overlap sequence length is 16 bp. Similarly, for Class 2,& D show that sequences fa and fb are complementary to sequences rb and ra respectively. In some embodiments, the optimized overlap sequence length for Class 2 is approximately 8 base pairs for each box (e.g., fa, fb, rb, ra, etc.).

Next, at block, the FastCloneAssist applicationcan generate a report. The report can comprise a notification, message, text file, image file, link, or other report which can be used to identify the primer designed by the FastCloneAssist application. According to various examples, the report can include a FASTA format of the final construct sequence, translations in all three frames, the designed primer sequences along with their respective melting temperatures and lengths, etc. In some embodiments, the FastCloneAssist applicationcan generate the report based at least in part on the selected melting temperature from block, the selected primer length from block, and/or the selected overlap sequences from block. In some embodiments, the FastCloneAssist applicationcan send the report to a client deviceto be displayed on a displayof the client device. After block, the flowchart ofends.